Inhibition by substrate of fructose 1,6-bisphosphatase purified from rat kidney cortex. Calculation of the kinetic constants of the enzyme

Fructose 1,6-bisphosphatase is a typical enzyme that is severely inhibited by its own substrate. This makes it difficult to determine all the parameters involved in its kinetics. It has been shown recently that if Vm is satisfactorily estimated the remaining parameters can be determined using the Hill plot (Bounias, M. (1988) Biochem. Int. 17, 147-154). The enzyme has been purified from rat kidney cortex nearly to homogeneity, and its kinetic constants have been calculated using a rigorous algebraic method. The most interesting result is that the substrate is unable to bind to the free enzyme as an inhibitor, which indicates that the enzyme lacks an allosteric site for hexose bisphosphates.     

The effect of pH on the cooperative behavior of aspartate transcarbamylase from Escherichia coli.

Saturation curves of activity versus concentration were determined for aspartate transcarbamylase from Escherichia coli (EC 2.1.3.2) for the substrate L-aspartate at saturating carbamyl phosphate (4.8 mM) in buffered solution at pH values from 6.0 to 12.0. Hill coefficients were obtained from the sigmoidal curves. At pH values from 7.8 to 9.1, where substrate inhibition causes difficulties in the Hill approximation, our kinetic scheme includes substrate inhibition and residual activity in the abortive enzyme-substrate complex. The plot of Hill coefficient versus pH has pKalpha values of 7.4 and 9.8 at the half-maximum positions of the curve which has a plateau from pH 8.1 to 9.1. These pKalpha values may be associated with functional groups involved in the allosteric transition which activates the enzyme. A plot of [S]0.5 versus pH shows a pKalpha of 8.5, which may belong to a residue either at or near the aspartate binding site. At 50 mM aspartate concentration the pH-rate profile shows maxima at pH values of 8.8 and 10.0 (cf. Weitzman, P.D.J., and Wilson, I.B.(1966)J. Biol. Chem. 2418 5481-5488, who used 100 mM aspartate). However, when the pH-dependent substrate inhibition is included, the calculated Vmax--H curve is bell-shaped like that of the isolated catalytic subunit.     

Evaluation of Hill slopes and Hill coefficients when the saturation binding or velocity is not known.

1. The Hill coefficient (nH), an often-used measure of deviations from hyperbolic behaviour (nonhyperbolicity) in kinetic and binding systems, is usually estimated from the maximum or minimum slope of the Hill plot. The method depends strongly on the assumed magnitude of the asymptotic velocity (V) or binding (P) whose evaluation may be difficult in nonlinear/co-operative systems. Therefore, alternative procedures were devised for the estimation nH which do not require the prior knowledge of V or P. 2. When pairs of velocity/binding readings (v and w) are obtained at concentrations of c and alpha c, respectively (where alpha is a fixed constant), then the relation between w and v is described by a hyperbola, provided that Hill's equation is valid. In this case, linearizing plots, v/w versus v, w versus, w/v, and 1/w versus 1/v, can be used for estimation of the degree of the equation. However, if the Hill expression is applicable, these methods are not efficient and traditional procedures, particularly nonlinear regression, should be used. 3. The 'linearizing' plots of the Hill equation can be applied advantageously for the evaluation of the Hill slope and of nH also in the general case, when the Hill expression is actually not valid, provided that deviations from hyperbolic behaviour are positive. Appropriately extrapolated intercepts of the first two plots estimate alphanH. Furthermore, the slope of the third plot yields, similarly to the method of Kurganov et al., a continuous measure of the Hill slope (including its maximum) at all concentrations. The agreement is, at positive nonhyperbolicities, excellent theoretical values of Hill slopes and coefficients and those estimated by the proposed methods. 4. A coefficient of nonhyperbolicity (theta) is defined for 2nd-degree rate equations which provides a quantitative measure of positive or negative deviation from first-degree, hyperbolic characteristics. It is closely related to the Hill coefficient.     

Influence of carbachol on the kinetic parameters of the contractile response to noradrenaline in the rat vas deferens

Graphic and mathematical analysis of kinetics of the rat vas deferens contractile response to noradrenaline showed that alpha1-adrenoceptors mediating the contraction were in different functional states. In some organs, these receptors were homogeneous with the Hill coefficient n = 1 (the linear mode of the Scatchard plot), in the others--not homogeneous, with n > 1 (not linear mode of the Scatchard plot). Carbachol increased the contractile response to noradrenaline. As in the control, two types of response were revealed: 1. The Hill coefficient n < 1 (biphasic mode of the Scatchard plot) with two pools (high and low affinity) of alpha1-adrenoceptors, and 2. The Hill coefficient n = 1 (the linear mode of the Scatchard plot). These results suggest that the influence of carbachol is caused by the local interaction of M-cholinergic and alpha1-adrenergic systems.     

pH-dependence of the triose phosphate isomerase reaction

1. Some kinetic properties of aspartate transcarbamoylase (EC 2.1.3.2), that had been purified approx. 20-fold from wheat germ, were studied. 2. A plot of enzyme activity against pH showed a low maximum at pH8.4 and a second, higher, maximum at pH10.5. A plot of percentage inhibition by 0.2mm-UMP against pH was approximately parallel to the plot of activity against pH, except that between pH6.5 and 7.5 the enzyme was insensitive to 0.2mm-UMP. 3. Kinetics were studied in detail at pH10.0 and 25 degrees C. In the absence of UMP, initial-rate plots were hyperbolic when the concentration of either substrate was varied. UMP decreased both V(max.) and K(m) in plots of initial rate against l-aspartate concentration, but the plots remained hyperbolic. However, UMP converted plots of initial rate against carbamoyl phosphate concentration into a sigmoidal shape, without significantly affecting V(max.). Plots of initial rate against UMP concentration were also sigmoidal. 4. The theoretical model proposed by Monod et al. (1965) gave a partial explanation of these results. When quasi-equilibrium conditions were assumed analysis in terms of this model suggested a trimeric enzyme binding the allosteric ligands, carbamoyl phosphate and UMP, nearly exclusively to the R and T conformational states respectively, and existing predominantly in the R state when ligands were absent. However, the values of the Hill coefficients for the co-operativity of each allosteric ligand were somewhat less than those predicted by the theory. 5. Some of the implications of these results are discussed, and the enzyme is contrasted with the well-known aspartate transcarbamoylase of Escherichia coli.     

Purification and properties of phosphofructokinase (EC 2.7.1.11) of Saccharomyces carlsbergensis.

A procedure for the purification of phosphofructokinase from brewer's yeast (Saccharomyces carlsbergensis) is reported. Treatments with organic solvents and heat were avoided and chromatographic and filtration techniques in the presence of phenylmethane sulfonyl fluoride were mainly used. The purified enzyme is homogeneous in disc gel electrophoresis and according to sedimentation velocity and equilibrium measurements in the ultracentrifuge. The isoelectric point determined by focusing was 5.3. Absorption spectra, fluorescence spectra and circular dichroism spectrum are given. The molecular weight of the purified enzyme determined by gel filtration was 720 000, in agreement with that of the enzyme in the raw extract. This confirms the results of sedimentation velocity experiments which gave a value of SO20, W equals 19.4. Alkaline treatment leads to a dissociation of the native enzyme, yielding an inactive species with a molecular weight of 360 000. In 6M guanidine hydrochloride the enzyme dissociates into subunits with a mean molecular weight of 90 000 as obtained by ultracentrifugation analysis. This suggests a structure composed of 8 monomers. The specific activity of the enzyme was 116 U/mg under optimum conditions. The enzyme activity was proportional to the enzyme concentration in the range of 6 times 10- minus 12 M to 3 times 10- minus 7 M. The Michaelis constants and Hill coefficients for fructose 6-phosphate and AMP, the pH optima, and the stability properties of the enzyme are reported. Furthermore, the activation energy is given and it is shown that under saturating conditions, a straight Arrhenius plot obtains, whereas the plot is discontinuous at high ATP concentrations and at pH 7.6.     

The role of arginine residues in the function of D-glyceraldehyde-3-phosphate dehydrogenase.

Inactivation of apo-glyceraldehyde-3-phosphate dehydrogenase from rat skeletal muscle in the presence of butanedione is the result of modification of one arginyl residue per subunit of the tetrameric enzyme molecule. The loss of activity follows pseudo-first-order kinetics. NAD+ increases the apparent first-order rate constant of inactivation. The effect of NAD+ on the enzyme inactivation is cooperative (Hill coefficient = 2.3--3.2). Glyceraldehyde 3-phosphate protected the holoenzyme against inactivation, decreasing the rate constant of the reaction. At saturating concentrations of substrate the protection was complete. The Hill plot demonstrates that the effect is cooperative. This suggests that subunit interactions in the tetrameric holoenzyme molecule may affect the reactivity of the essential arginyl residues. In contrast, glyceraldehyde 3-phosphate had no effect on the rate of inactivation of the apoenzyme in the presence of butanedione. 100 mM inorganic phosphate protected both the apoenzyme and holoenzyme against inactivation. The involvement of the microenvironment of the arginyl residues in the functionally important conformational changes of the enzyme is discussed.     

Regulation of Azotobacter chroococcum invertase

Synthesis of the Azotobacter chroococcum invertase was found to be dependent on sucrose or raffinose in the growth medium. The activity of this invertase was slightly inhibited by glucose. Fructose, which by itself did not affect the enzyme activity, protected invertase from glucose inhibition. Per cent residual activity plotted against glucose concentration, and Hill plot indicated that this monosaccharide binds to one interacting site of the enzyme. The results show that regulation of this prokaryotic enzyme clearly differs from that of eukaryotic orgnisms.     

Purification and some properties of the secreted arginase of the lichen Evernia prunastri and its regulation by usnic acid

A secreted argnase (molecular weight 245 000) from Evernia prunastri thallus has been purified 1480-fold from media in which the thalli were incubated for 8 h in the dark. The enzyme is a glycoprotein which contains 280 residues of glucose, 27 of fructose and 85 of mannose per molecule. The K_m-value of the enzyme has been estimated as 1.5 mM for L-arginine, with an interaction coefficient of n_H ≅ 1, calculated from Hill plot. The enzyme is activated by D-usnic acid, the only phenol which appears in the incubation media. This phenol behaves as a non-essential activator of the enzyme with a K_a-value of 0.19 mM.     

A new measure of cooperativity in protein-ligand binding

An allosteric binding system consisting of a single ligand and a nondissociating macromolecule having multiple binding sites can be represented by a binding polynomial. Various properties of the binding process can be obtained by analyzing the coefficients of the binding polynomial and such functions as the binding curve and the Hill plot. The Hill plot has an asymptote of unit slope at each end and the departure of the slope from unity at any point can be used to measure the effective interaction free energy at that point. Of particular interest in detecting and measuring cooperativity are extrema of the Hill slope and its value at the half-saturation point. If the binding polynomial is symmetric, then there is an extremum of the Hill slope at the half-saturation point. This value, the Hill coefficient, is a convenient measure of cooperativity. The purpose of this paper is to express the Hill coefficient for symmetric binding polynomials in terms of the roots of the polynomial and to give an interpretation of cooperativity in terms of the geometric pattern of the roots in the complex plane. This interpretation is then applied to the binding polynomials for the MWC (Monod-Wyman-Changeux) and KNF (Koshland-Nemethy-Filmer) models.     

Carbamate kinase from Pseudomonas aeruginosa: purification, characterization, physiological role, and regulation.

Pseudomonas aeruginosa PAO1 possessed a carbamate kinase (CKase) distinct from carbamoylphosphate synthetase as well as from a constitutive acetate kinase which also catalyzes the phosphorylation of ADP by carbamoylphosphate. CKase was purified to homogeneity. Polyacrylamide gel electrophoresis of cross-linked CKase in the presence of sodium dodecyl sulfate showed that the enzyme consists of two subunits with identical molecular weights (37,000). The optimal pH of enzyme activity is 7.0. The double-reciprocal plot for carbamoylphosphate was linear at 2 mM ADP, yielding an apparent Km of 5 mM. However, at 0.25 mM ADP, the plot was concave upward, and a Hill plot of the data yielded a coefficient of 1.4. This apparent cooperativity at low ADP concentrations might serve to reduce the extent of catabolism of carbamoylphosphate under growth conditions yielding high energy charge. Experiments on the regulation of synthesis under various growth conditions showed a response to three regulatory signals: CKase was induced to high levels by anaerobiosis, induced to moderate levels by arginine, and repressed by ammonia. Thus, CKase expression is regulated in a manner that allows the enzyme to function as a provider of ammonia under aerobic conditions and of ATP under anaerobic conditions. ATP was an effective inhibitor of CKase activity; this inhibition provides the cell with an effective mechanism for avoiding a futile cycle resulting from the simultaneous operation of CKase and carbamoylphosphate synthetase when cells are grown in the presence of exogenous arginine.     

Regulation of Chorismate mutase-prephenate dehydratase and prephenate dehydrogenase from alcaligenes eutrophus.

Highly purified enzymes from Alcaligenes eutrophus H 16 were used for kinetic studies. Chorismate mutase was feedback inhibited by phenylalanine. In the absence of the inhibitor, the double-reciprocal plot was linear, yielding a Km for chorismate of 0.2 mM. When phenylalanine was present, a pronounced deviation from the Michaelis-Menten hyperbola occurred. The Hill coefficient (n) was 1.7, and Hill plots of velocity versus inhibitor concentrations resulted in a value of n' = 2.3, indicating positive cooperativity. Chorismate mutase was also inhibited by prephenate, which caused downward double-reciprocal plots and a Hill coefficient of n = 0.7, evidence for negative cooperativity. The pH optimum of chorismate mutase ranged from 7.8 to 8.2; its temperature optimum was 47 C. Prephenate dehydratase was competitively inhibited by phenylalanine and activated by tyrosine. Tyrosine stimulated its activity up to 10-fold and decreased the Km for prephenate, which was 0.67 mM without effectors. Tryptophan inhibited the enzyme competitively. Its inhibition constant (Ki = 23 muM) was almost 10-fold higher than that determined for phenylalanine (Ki = 2.6 muM). The pH optimum of prephenate dehydratase was pH 5.7; the temperature optimum was 48 C. Prephenate dehydrogenase was feedback inhibited by tyrosine. Inhibition was competitive with prephenate (Ki = 0.06 mM) and noncompetitive with nicotinamide adenine dinucleotide. The enzyme was further subject to product inhibition by p-hydroxyphenylpyruvate (Ki = 0.13 mM). Its Km for prephenate was 0.045 mM, and that for nicotinamide adenine dinucleotide was 0.14 mM. The pH optimum ranged between 7.0 and 7.6; the temperature optimum was 38 C. It is shown how the sensitive regulation of the entire enzyme system leads to a well-balanced amino acid production.     

Site-specific modification of Escherichia coli DNA polymerase I large fragment with pyridoxal 5'-phosphate

Pyridoxal 5'-phosphate (PLP) is an inhibitor of DNA polymerase activity of Escherichia coli DNA polymerase I large fragment. Kinetic studies indicated that overall PLP inhibition was noncompetitive with respect to dNTP, and Hill plot analysis revealed that two molecules of PLP were involved in the inhibition. Reduction of the PLP-treated enzyme with sodium [3H]borohydride resulted in covalent incorporation of 3 mol of PLP/mol of enzyme. This incorporation was at lysine residues exclusively, and the PLP-modified enzyme was not capable of DNA polymerase activity. The presence of dNTP during the modification reaction blocked the incorporation of 1 mol of PLP/mol of enzyme. Similar results were obtained in the presence or absence of template-primer. These data indicate that a PLP target lysine is in or around a dNTP binding site that is essential for polymerase activity and that this binding site is functional in the absence of template-primer. The enzyme modified in the presence of dNTP, containing 2 mol of PLP/mol of enzyme, was capable of DNA polymerase activity but was unable to conduct elongation of product molecules beyond a short oligonucleotide length.     

The convenience of the use of allosteric "probes" for the study of lipid--protein interactions in biological membranes: thermodynamic considerations.

One of the most commonly used methods to study enzyme (protein)-structure interactions at a more specific level is the use of the Arrhenius plots to detect the influence of the “environment” on the enzyme. We want to point out here that the use of a suitable “allosteric enzyme” would be a more sensitive method to detect influences of the environment than the study of Arrhenius plots. According to simple thermodynamic considerations, a change of more than 2.8 kcal/mol in the interaction between enzyme and membrane would be needed to give a noticeable change in the position of the break (T_i) of the corresponding Arrhenius plot. On the other hand, feeble changes in the membrane-enzyme interaction, of the order of 0·7–0·8 kcal/mol, would be enough to give a significant change in the values of n (Hill coefficient).     

Absence of positive co-operativity in the binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin to its cytosolic receptor protein.

The role of positive co-operativity in stabilizing the binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to the rat hepatic cytosolic TCDD receptor protein (Ah receptor) was investigated. The binding mechanism of TCDD was determined by kinetic means through equilibrium and saturation binding studies, and Scatchard and Hill plot analysis. In all studies, the slope of the Hill plot was close to 1.0, indicating the absence of positive co-operativity. Interpretation of the Scatchard plot was however complicated by the fact that both linear and nonlinear plots were experimentally obtained. The nonlinearity was shown to be an experimental artifact and a consequence not of co-operativity, but of high levels of nonspecific binding. The high level of nonspecific binding could be attributed to: (1) lipophilicity of the TCDD ligand, and (2) inefficient competition of receptor-bound [3H]TCDD. When nonspecific binding was minimized, the Scatchard slope was linear and in agreement with the Hill coefficient, thus indicating the lack of positive co-operativity in the binding of TCDD to the Ah receptor.     

Quantitative analysis of ultrasensitive responses

Ultrasensitive responses are common in cellular information transfer because they allow cells to decode extracellular stimuli in an all-or-none manner. Biochemical responses are usually analyzed by fitting the Hill equation, and the estimated Hill coefficient is taken as a measure of sensitivity. However, this approach is not appropriate if the response under consideration significantly deviates from the best-fit Hill equation. In addition, Hill coefficients greater than unity do not necessarily imply ultrasensitive behaviour if basal activation is significant. In order to circumvent these problems we propose a general method for the quantitative analysis of sensitivity, the relative amplification plot, which is based on the response coefficient defined in metabolic control analysis. To quantify sensitivity globally (i.e. over the whole stimulus range) we introduce the integral-based relative amplification coefficient. Our relative amplification approach can easily be extended to monotonically decreasing, bell-shaped or nonsaturated responses.     

Purification and characterization of pyruvate decarboxylase from sweet potato roots.

Pyruvate decarboxylase [2-oxo acid carboxy-lyase, EC 4.1.1.1] was isolated from sweet potato roots and was partially purified from healthy and diseased tissues. There was no appreciable difference in properties between the enzymes from healthy and diseased tissues. The molecular weight of the enzyme was found to be 240,000 by polyacrylamide gel electrophoresis. Since sodium dodecyl sulfate polyacrylamide gel electrophoresis gave a molecular weight of 60,000 for the monomeric form of the enzyme, it is likely that sweet potato pyruvate decarboxylase contains 4 single polypeptide chains. The optimal pH of the decarboxylation reaction was 6.1--6.6. The Lineweaver-Burk double reciprocal plot curved upward, and the Hill coefficient was more than 1, with low concentrations of pyruvate. The enzyme was localized in the cytosol fraction. The activity of the enzyme increased in response to black-rot fungus infection, but decreased in response to cutting.     

Beta-oxidation as channeled reaction linked to citric acid cycle: evidence from measurements of mitochondrial pyruvate oxidation during fatty acid degradation.

1. The kinetics of mitochondrial mammalian pyruvate dehydrogenase multienzyme complex (PDHC) is studied by the formation of CO2 using tracer amounts of [1-14C]pyruvate. It is found that the Hill plot results in a (pseudo-)cooperativity with a transition of n-1----3 at a pyruvate concentration about Ks. 2. Addition of L-carnitine, octanoate, palmitoyl-CoA or palmitate + L-carnitine + fatty acid-binding protein results in a Hill coefficient of n = 2 following the kinetics of pyruvate oxidation. 3. Addition of fatty acid-binding protein to an assay system oxidizing palmitate in presence of L-carnitine alters the pattern of the kinetics in the Hill plot so that an apparently lower level of L-carnitine is necessary for the reaction course of beta-degradation. 4. It is concluded that beta-degradation is a coordinated, multienzyme-complex based mechanism tightly linked to citric acid cycle and it is proposed that L-carnitine is actively involved into the reaction and not only functioning as carrier-molecule for transmembrane transport.